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1.  Dissection of the Role of Pili and Type 2 and 3 Secretion Systems in Adherence and Biofilm Formation of an Atypical Enteropathogenic Escherichia coli Strain 
Infection and Immunity  2013;81(10):3793-3802.
Atypical enteropathogenic Escherichia coli (aEPEC) strains are diarrheal pathogens that lack bundle-forming pilus production but possess the virulence-associated locus of enterocyte effacement. aEPEC strain 1551-2 produces localized adherence (LA) on HeLa cells; however, its isogenic intimin (eae) mutant produces a diffuse-adherence (DA) pattern. In this study, we aimed to identify the DA-associated adhesin of the 1551-2 eae mutant. Electron microscopy of 1551-2 identified rigid rod-like pili composed of an 18-kDa protein, which was identified as the major pilin subunit of type 1 pilus (T1P) by mass spectrometry analysis. Deletion of fimA in 1551-2 affected biofilm formation but had no effect on adherence properties. Analysis of secreted proteins in supernatants of this strain identified a 150-kDa protein corresponding to SslE, a type 2 secreted protein that was recently reported to be involved in biofilm formation of rabbit and human EPEC strains. However, neither adherence nor biofilm formation was affected in a 1551-2 sslE mutant. We then investigated the role of the EspA filament associated with the type 3 secretion system (T3SS) in DA by generating a double eae espA mutant. This strain was no longer adherent, strongly suggesting that the T3SS translocon is the DA adhesin. In agreement with these results, specific anti-EspA antibodies blocked adherence of the 1551-2 eae mutant. Our data support a role for intimin in LA, for the T3SS translocon in DA, and for T1P in biofilm formation, all of which may act in concert to facilitate host intestinal colonization by aEPEC strains.
PMCID: PMC3811761  PMID: 23897608
2.  Multi-functional analysis of Klebsiella pneumoniae fimbrial types in adherence and biofilm formation 
Virulence  2013;4(2):129-138.
Klebsiella pneumoniae is an opportunistic pathogen frequently associated with nosocomially acquired infections. Host cell adherence and biofilm formation of K. pneumoniae isolates is mediated by type 1 (T1P) and type 3 (MR/K) pili whose major fimbrial subunits are encoded by the fimA and mrkA genes, respectively. The E. coli common pilus (ECP) is an adhesive structure produced by all E. coli pathogroups and a homolog of the ecpABCDE operon is present in the K. pneumoniae genome. In this study, we aimed to determine the prevalence of these three fimbrial genes among a collection of 69 clinical and environmental K. pneumoniae strains and to establish a correlation with fimbrial production during cell adherence and biofilm formation. The PCR-based survey demonstrated that 96% of the K. pneumoniae strains contained ecpA and 94% of these strains produced ECP during adhesion to cultured epithelial cells. Eighty percent of the strains forming biofilms on glass produced ECP, suggesting that ECP is required, at least in vitro, for expression of these phenotypes. The fim operon was found in 100% of the strains and T1P was detected in 96% of these strains. While all the strains examined contained mrkA, only 57% of them produced MR/K fimbriae, alone or together with ECP. In summary, this study highlights the ability of K. pneumoniae strains to produce ECP, which may represent a new important adhesive structure of this organism. Further, it defines the multi-fimbrial nature of the interaction of this nosocomial pathogen with host epithelial cells and inert surfaces.
PMCID: PMC3654611  PMID: 23302788
adherence; Klebsiella pneumoniae; biofilms; K. oxytoca; E. coli common pilus
3.  Evaluation of the Prevalence and Production of Escherichia coli Common Pilus among Avian Pathogenic E. coli and Its Role in Virulence 
PLoS ONE  2014;9(1):e86565.
Avian pathogenic Escherichia coli (APEC) strains cause systemic and localized infections in poultry, jointly termed colibacillosis. Avian colibacillosis is responsible for significant economic losses to the poultry industry due to disease treatment, decrease in growth rate and egg production, and mortality. APEC are also considered a potential zoonotic risk for humans. Fully elucidating the virulence and zoonotic potential of APEC is key for designing successful strategies against their infections and their transmission. Herein, we investigated the prevalence of a newly discovered E. coli common pilus (ECP) for the subunit protein of the ECP pilus (ecpA) and ECP expression amongst APEC strains as well as the role of ECP in virulence. A PCR-based ecpA survey of a collection of 167 APEC strains has shown that 76% (127/167) were ecpA+. An immunofluorescence assay using anti-EcpA antibodies, revealed that among the ecpA+ strains, 37.8% (48/127) expressed ECP when grown in DMEM +0.5% Mannose in contact with HeLa cells at 37°C and/or in biofilm at 28°C; 35.4% (17/48) expressed ECP in both conditions and 64.6% (31/48) expressed ECP in biofilm only. We determined that the ecp operon in the APEC strain χ7122 (ecpA+, ECP-) was not truncated; the failure to detect ECP in some strains possessing non-truncated ecp genes might be attributed to differential regulatory mechanisms between strains that respond to specific environmental signals. To evaluate the role of ECP in the virulence of APEC, we generated ecpA and/or ecpD-deficient mutants from the strain χ7503 (ecpA+, ECP+). Deletion of ecpA and/or ecpD abolished ECP synthesis and expression, and reduced biofilm formation and motility in vitro and virulence in vivo. All together our data show that ecpA is highly prevalent among APEC isolates and its expression could be differentially regulated in these strains, and that ECP plays a role in the virulence of APEC.
PMCID: PMC3900561  PMID: 24466152
4.  A Toxin-Antitoxin Module of Salmonella Promotes Virulence in Mice 
PLoS Pathogens  2013;9(12):e1003827.
Toxin-antitoxin (TA) modules are widely prevalent in both bacteria and archaea. Originally described as stabilizing elements of plasmids, TA modules are also widespread on bacterial chromosomes. These modules promote bacterial persistence in response to specific environmental stresses. So far, the possibility that TA modules could be involved in bacterial virulence has been largely neglected, but recent comparative genomic studies have shown that the presence of TA modules is significantly associated with the pathogenicity of bacteria. Using Salmonella as a model, we investigated whether TA modules help bacteria to overcome the stress conditions encountered during colonization, thereby supporting virulence in the host. By bioinformatics analyses, we found that the genome of the pathogenic bacterium Salmonella Typhimurium encodes at least 11 type II TA modules. Several of these are conserved in other pathogenic strains but absent from non-pathogenic species indicating that certain TA modules might play a role in Salmonella pathogenicity. We show that one TA module, hereafter referred to as sehAB, plays a transient role in virulence in perorally inoculated mice. The use of a transcriptional reporter demonstrated that bacteria in which sehAB is strongly activated are predominantly localized in the mesenteric lymph nodes. In addition, sehAB was shown to be important for the survival of Salmonella in these peripheral lymphoid organs. These data indicate that the transient activation of a type II TA module can bring a selective advantage favouring virulence and demonstrate that TA modules are engaged in Salmonella pathogenesis.
PMCID: PMC3868539  PMID: 24385907
5.  SOD2 gene Val16Ala polymorphism is associated with macroalbuminuria in Mexican Type 2 Diabetes patients: a comparative study and meta-analysis 
BMC Medical Genetics  2013;14:110.
Several studies in type 2 diabetes patients have shown significant associations between the SOD2 gene Val16Ala polymorphism and albuminuria, but this association has not been explored in the Mexican population.
We evaluated the association between the SOD2 gene Val16Ala polymorphism (rs4880) and macroalbuminuria in a sample of 994 unrelated Mexican type 2 diabetes patients. The study included 119 subjects with urinary albumin >300 mg/dL and 875 subjects with urinary albumin ≤ 30 mg/dL. Genotyping of the SOD2 gene Val16Ala SNP was carried out with Real-Time Polymerase Chain Reaction (RT-PCR).
The frequency of the TT genotype was 6.7% higher in participants with macroalbuminuria than in the normoalbuminuria group (16.8% vs. 10.1%). Using a logistic regression analysis, we observed that individuals with the CC genotype had significantly lower risks of macroalbuminuria than those with the TT genotype (OR=0.42, p=0.034). We carried out a meta-analysis combining our data with data from four previous studies and estimated an odds ratio (95% CI) for the C allele (with respect to the reference T allele) of 0.65 (0.52-0.80, p<0.001).
A significant association was found between the SOD2 Val16Ala polymorphism and macroalbuminuria in a sample of Mexican type 2 diabetes patients.
PMCID: PMC3853578  PMID: 24119114
Mexicans; Macroalbuminuria; SOD2; Type 2 diabetes; Val16Ala polymorphism
6.  The Interaction between Factor H and Von Willebrand Factor 
PLoS ONE  2013;8(8):e73715.
Complement factor H (fH) is a plasma protein that regulates activation of the alternative pathway, and mutations in fH are associated with a rare form of thrombotic microangiopathy (TMA), known as atypical hemolytic uremic syndrome (aHUS). A more common TMA is thrombotic thrombocytopenic purpura, which is caused by the lack of normal ADAMTS-13-mediated cleavage of von Willebrand factor (VWF). We investigated whether fH interacts with VWF and affects cleavage of VWF. We found that factor H binds to VWF in plasma, to plasma-purified VWF, and to recombinant A1 and A2 domains of VWF as detected by co-immunoprecipitation (co-IP) and surface plasmon resonance assays. Factor H enhanced ADAMTS-13-mediated cleavage of recombinant VWF-A2 as determined by quantifying the cleavage products using Western-blotting, enhanced cleavage of a commercially available fragment of VWF-A2 (FRETS-VWF73) as determined by fluorometric assay, and enhanced cleavage of ultralarge (UL) VWF under flow conditions as determined by cleavage of VWF-platelet strings attached to histamine stimulated endothelial cells. Using recombinant full-length and truncated fH molecules, we found that the presence of the C-terminal half of fH molecule is important for binding to VWF-A2 and for enhancing cleavage of the A2 domain by ADAMTS-13. We conclude that factor H binds to VWF and may modulate cleavage of VWF by ADAMTS-13.
PMCID: PMC3753316  PMID: 23991205
7.  Analysis of the contribution of FTO, NPC1, ENPP1, NEGR1, GNPDA2 and MC4R genes to obesity in Mexican children 
BMC Medical Genetics  2013;14:21.
Recent genome wide association studies (GWAS) and previous positional linkage studies have identified more than 50 single nucleotide polymorphisms (SNPs) associated with obesity, mostly in Europeans. We aimed to assess the contribution of some of these SNPs to obesity risk and to the variation of related metabolic traits, in Mexican children.
The association of six European obesity-related SNPs in or near FTO, NPC1, ENPP1, NEGR1, GNPDA2 and MC4R genes with risk of obesity was tested in 1,463 school-aged Mexican children (Ncases = 514; Ncontrols = 949). We also assessed effects of these SNPs on the variation of body mass index (BMI), fasting serum insulin levels, fasting plasma glucose levels, total cholesterol and triglyceride levels, in a subset of 1,171 nonobese Mexican children.
We found a significant effect of GNPDA2 rs10938397 on risk of obesity (odds ratio [OR] = 1.30; P = 1.34 × 10-3). Furthermore, we found nominal associations between obesity risk or BMI variation and the following SNPs: ENPP1 rs7754561, MC4R rs17782313 and NEGR1 rs2815752. Importantly, the at-risk alleles of both MC4R rs17782313 and NPC1 rs1805081 showed significant effect on increased fasting glucose levels (β = 0.36 mmol/L; P = 1.47 × 10-3) and decreased fasting serum insulin levels (β = −0.10 μU/mL; P = 1.21 × 10-3), respectively.
Our present results suggest that some obesity-associated SNPs previously reported in Europeans also associate with risk of obesity, or metabolic quantitative traits, in Mexican children. Importantly, we found new associations between MC4R and fasting glucose levels, and between NPC1 and fasting insulin levels.
PMCID: PMC3577489  PMID: 23375129
Obesity; Mexican children; Single nucleotide polymorphism
8.  Adiponectin in eutrophic and obese children as a biomarker to predict metabolic syndrome and each of its components 
BMC Public Health  2013;13:88.
Obesity is associated with the rise of noncommunicable diseases worldwide. The pathophysiology behind this disease involves the increase of adipose tissue, being inversely related to adiponectin, but directly related to insulin resistance and metabolic syndrome (MetS). Therefore, this study aimed to determine the relationship between adiponectin levels with each component of MetS in eutrophic and obese Mexican children.
A cross sectional study was conducted in 190 school-age children classified as obese and 196 classified as eutrophic. Adiponectin, glucose, insulin, high density lipoprotein cholesterol (HDL-C) and triglycerides were determined from a fasting blood sample. Height, weight, waist circumference, systolic and diastolic blood pressures (BP) were measured; MetS was evaluated with the IDF definition. The study groups were divided according to tertiles of adiponectin, using the higher concentration as a reference. Linear regression analysis was used to assess the association between adiponectin and components of the MetS. Finally, stepwise forward multiple logistic regression analysis controlling for age, gender, basal HOMA-IR values and BMI was performed to determine the odds ratio of developing MetS according to adiponectin tertiles.
Anthropometric and metabolic measurements were statistically different between eutrophic and obese children with and without MetS (P <0.001). The prevalence of MetS in obese populations was 13%. Adiponectin concentrations were 15.5 ± 6.1, 12.0 ± 4.8, 12.4 ± 4.9 and 9.4 ± 2.8 μg/mL for eutrophic and obese subjects, obese without MetS, and obese with MetS, respectively (P <0.001). Obese children with low values of adiponectin exhibited a higher frequency of MetS components: abdominal obesity, 49%; high systolic BP, 3%; high diastolic BP, 2%; impaired fasting glucose, 17%; hypertriglyceridemia, 31%; and low HDL-C values, 42%. Adjusted odds ratio of presenting MetS according to adiponectin categories was 10.9 (95% CI 2.05; 48.16) when the first tertile was compared with the third.
In this sample of eutrophic and obese Mexican children we found that adiponectin concentrations and MetS components have an inversely proportional relationship, which supports the idea that this hormone could be a biomarker for identifying individuals with risk of developing MetS.
PMCID: PMC3570482  PMID: 23363707
Obesity; Adiponectin; Child; Insulin resistance; Metabolic syndrome; Biomarker
9.  Reconstitution of Platelet Glycoprotein Ib-IX Complex in Phospholipid Bilayer Nanodiscs† 
Biochemistry  2011;50(49):10598-10606.
Glycoprotein (GP)Ib-IX complex expressed on platelet plasma membrane is involved in thrombosis and hemostasis by initiating platelet adhesion to von Willebrand factor (VWF) exposed at the injured vessel wall. While most of the knowledge for GPIb-IX is obtained from studies on platelets and transfected mammalian cells expressing GPIb-IX, there is not an in vitro membrane system that allows systematic analysis of this receptor. The phospholipid bilayer Nanodisc composed of a patch of phospholipid surrounded by membrane scaffold protein is an attractive tool for membrane protein study. We show here that GPIb-IX purified from human platelets has been reconstituted into the Nanodisc. Nanodisc-reconstituted GPIb-IX was able to bind various conformation-sensitive monoclonal antibodies. Furthermore, it bound to VWF in the presence of botrocetin with an apparent Kd of 0.73 ± 0.07 nM. The binding to VWF was inhibited by anti-GPIbα antibodies with epitopes overlapping with the VWF-binding site, but not by anti-GPIbβ monoclonal antibody RAM.1. Finally, Nanodisc-reconstituted GPIb-IX exhibited similar ligand-binding activity as the isolated extracellular domain of GPIbα. In conclusion, GPIb-IX in Nanodiscs adopts native-like conformation and possesses the ability to bind its natural ligands, thus making Nanodisc a suitable in vitro platform for further investigation on this hemostatically important receptor complex.
PMCID: PMC3552449  PMID: 22080766
10.  The SNP at −592 of human IL-10 gene is associated with serum IL-10 levels and increased risk for human papillomavirus cervical lesion development 
Women with Human Papilloma Virus (HPV) persistence are characterized by high levels of IL-10 at cervix. We have determined whether polymorphisms of IL-10 gene promoter might be associated with increased risk of squamous intraepithelial cervical lesions (SICL) and whether exist significative differences of IL-10 mRNA expression at cervix and systemic and serum IL-10 protein between SICL cases and non-Cervical Lesions (NCL).
Peripheral blood samples from SICL (n = 204) and NCL (n = 166) were used to detect IL-10 promoter polymorphisms at loci -592A/C (rs1800872), -819C/T (rs1800871), -1082A/G (rs1800896), -1352A/G (rs1800893), by allelic discrimination and to evaluate serum IL-10 protein. Cervical epithelial scrapings from NCL and biopsies from SICLs were used for HPV-typing and to evaluate IL-10 mRNA expression level. The systemic and local IL-10 mRNA expression levels were measured by real time-PCR. Genotypic and allelic frequencies of the selected polymorphisms were analyzed by logistic regression, adjusting by age and HPV-genotype, to determine the association with SICL.
No significant differences were found between genotype frequencies at loci −819, -1082, and −1352. Individuals carrying at least one copy of risk allele A of polymorphism −592 had a two-fold increased risk of developing SICL [adjusted odds ratio (OR), 2.02 (95% CI, 1.26-3.25), p = 0.003], compared to NCL. The IL-10 mRNA expression and serum IL-10 protein, were significantly higher in SICL cases (p < 0.01), being higher in patients carrying the risk allele A.
The −592 polymorphism is associated with increased risk of SICL and can serve as a marker of genetic susceptibility to SICL among Mexican women. According to IL-10 levels found in SICL, IL-10 can be relevant factor for viral persistence and progression disease.
PMCID: PMC3552694  PMID: 23148667
IL-10 promoter polymorphisms; Squamous intraepithelial cervical lesions; IL-10 expression; Risk factors
11.  Dyslipidemia in a Cohort of HIV-infected Latin American Children Receiving Highly Active Antiretroviral Therapy* 
Journal of Tropical Pediatrics  2010;57(5):324-332.
In order to describe the prevalence of hypercholesterolemia and hypertriglyceridemia in a cohort of HIV-infected children and adolescents in Latin America and to determine associations with highly active antiretroviral therapy (HAART), we performed this cross-sectional analysis within the NICHD International Site Development Initiative pediatric cohort study. Eligible children had to be at least 2 years of age and be on HAART. Among the 477 eligible HIV-infected youth, 98 (20.5%) had hypercholesterolemia and 140 (29.4%) had hypertriglyceridemia. In multivariable analyses, children receiving protease inhibitor (PI)-containing HAART were at increased risk for hypercholesterolemia [adjusted odds ratio (AOR) = 2.7, 95% confidence interval (CI) 1.3–5.6] and hypertriglyceridemia (AOR = 3.5, 95% CI 1.9–6.4) compared with children receiving non-nucleoside reverse transcriptase inhibitor (NNRTI)-containing HAART. In conclusion, HIV-infected youth receiving PI-containing HAART in this Latin American cohort were at increased risk for hypercholesterolemia and hypertriglyceridemia compared with those receiving NNRTI-containing HAART.
PMCID: PMC3203396  PMID: 20889625
HIV; cholesterol; triglycerides; pediatric
12.  Haplotypes in the CRP Gene Associated with Increased BMI and Levels of CRP in Subjects with Type 2 Diabetes or Obesity from Southwestern Mexico 
Experimental Diabetes Research  2012;2012:982683.
Objective. We evaluated the association between four polymorphisms in the CRP gene with circulating levels of C-reactive protein (CRP), type 2 diabetes (T2D), obesity, and risk score of coronary heart disease. Methods. We studied 402 individuals and classified them into four groups: healthy, obese, T2D obese, and T2D without obesity, from Guerrero, Southwestern Mexico. Blood levels of CRP, glucose, cholesterol, triglycerides, and leukocytes were measured. Genotyping was performed by PCR/RFLP, and the risk score for coronary heart disease was determined by the Framingham's methodology. Results. The TT genotype of SNP rs1130864 was associated with increased body mass index and T2D patients with obesity. We found that the haplotype 2 (TGAG) was associated with increased levels of CRP (β = 0.3; 95%CI: 0.1, 0.5; P = 0.005) and haplotype 7 (TGGG) with higher body mass index (BMI) (β = 0.2; 95%CI: 0.1, 0.3; P < 0.001). The risk score for coronary heart disease was associated with increased levels of CRP, but not with any polymorphism or haplotype. Conclusions. The association between the TT genotype of SNP rs1130864 with obesity and the haplotype 7 with BMI may explain how obesity and genetic predisposition increase the risk of diseases such as T2D in the population of Southwestern Mexico.
PMCID: PMC3463182  PMID: 23049543
13.  Evaluation of the imputation performance of the program IMPUTE in an admixed sample from Mexico City using several model designs 
BMC Medical Genomics  2012;5:12.
We explored the imputation performance of the program IMPUTE in an admixed sample from Mexico City. The following issues were evaluated: (a) the impact of different reference panels (HapMap vs. 1000 Genomes) on imputation; (b) potential differences in imputation performance between single-step vs. two-step (phasing and imputation) approaches; (c) the effect of different INFO score thresholds on imputation performance and (d) imputation performance in common vs. rare markers.
The sample from Mexico City comprised 1,310 individuals genotyped with the Affymetrix 5.0 array. We randomly masked 5% of the markers directly genotyped on chromosome 12 (n = 1,046) and compared the imputed genotypes with the microarray genotype calls. Imputation was carried out with the program IMPUTE. The concordance rates between the imputed and observed genotypes were used as a measure of imputation accuracy and the proportion of non-missing genotypes as a measure of imputation efficacy.
The single-step imputation approach produced slightly higher concordance rates than the two-step strategy (99.1% vs. 98.4% when using the HapMap phase II combined panel), but at the expense of a lower proportion of non-missing genotypes (85.5% vs. 90.1%). The 1,000 Genomes reference sample produced similar concordance rates to the HapMap phase II panel (98.4% for both datasets, using the two-step strategy). However, the 1000 Genomes reference sample increased substantially the proportion of non-missing genotypes (94.7% vs. 90.1%). Rare variants (<1%) had lower imputation accuracy and efficacy than common markers.
The program IMPUTE had an excellent imputation performance for common alleles in an admixed sample from Mexico City, which has primarily Native American (62%) and European (33%) contributions. Genotype concordances were higher than 98.4% using all the imputation strategies, in spite of the fact that no Native American samples are present in the HapMap and 1000 Genomes reference panels. The best balance of imputation accuracy and efficiency was obtained with the 1,000 Genomes panel. Rare variants were not captured effectively by any of the available panels, emphasizing the need to be cautious in the interpretation of association results for imputed rare variants.
PMCID: PMC3436779  PMID: 22549150
14.  Development of a Panel of Genome-Wide Ancestry Informative Markers to Study Admixture Throughout the Americas 
PLoS Genetics  2012;8(3):e1002554.
Most individuals throughout the Americas are admixed descendants of Native American, European, and African ancestors. Complex historical factors have resulted in varying proportions of ancestral contributions between individuals within and among ethnic groups. We developed a panel of 446 ancestry informative markers (AIMs) optimized to estimate ancestral proportions in individuals and populations throughout Latin America. We used genome-wide data from 953 individuals from diverse African, European, and Native American populations to select AIMs optimized for each of the three main continental populations that form the basis of modern Latin American populations. We selected markers on the basis of locus-specific branch length to be informative, well distributed throughout the genome, capable of being genotyped on widely available commercial platforms, and applicable throughout the Americas by minimizing within-continent heterogeneity. We then validated the panel in samples from four admixed populations by comparing ancestry estimates based on the AIMs panel to estimates based on genome-wide association study (GWAS) data. The panel provided balanced discriminatory power among the three ancestral populations and accurate estimates of individual ancestry proportions (R2>0.9 for ancestral components with significant between-subject variance). Finally, we genotyped samples from 18 populations from Latin America using the AIMs panel and estimated variability in ancestry within and between these populations. This panel and its reference genotype information will be useful resources to explore population history of admixture in Latin America and to correct for the potential effects of population stratification in admixed samples in the region.
Author Summary
Individuals from Latin America are descendants of multiple ancestral populations, primarily Native American, European, and African ancestors. The relative proportions of these ancestries can be estimated using genetic markers, known as ancestry informative markers (AIMs), whose allele frequency varies between the ancestral groups. Once determined, these ancestral proportions can be correlated with normal phenotypes, can be associated with disease, can be used to control for confounding due to population stratification, or can inform on the history of admixture in a population. In this study, we identified a panel of AIMs relevant to Latin American populations, validated the panel by comparing estimates of ancestry using the panel to ancestry determined from genome-wide data, and tested the panel in a diverse set of populations from the Americas. The panel of AIMs produces ancestry estimates that are highly accurate and appropriately controlled for population stratification, and it was used to genotype 18 populations from throughout Latin America. We have made the panel of AIMs available to any researcher interested in estimating ancestral proportions for populations from the Americas.
PMCID: PMC3297575  PMID: 22412386
15.  Tetrodotoxin (TTX) as a Therapeutic Agent for Pain 
Marine Drugs  2012;10(2):281-305.
Tetrodotoxin (TTX) is a potent neurotoxin that blocks voltage-gated sodium channels (VGSCs). VGSCs play a critical role in neuronal function under both physiological and pathological conditions. TTX has been extensively used to functionally characterize VGSCs, which can be classified as TTX-sensitive or TTX-resistant channels according to their sensitivity to this toxin. Alterations in the expression and/or function of some specific TTX-sensitive VGSCs have been implicated in a number of chronic pain conditions. The administration of TTX at doses below those that interfere with the generation and conduction of action potentials in normal (non-injured) nerves has been used in humans and experimental animals under different pain conditions. These data indicate a role for TTX as a potential therapeutic agent for pain. This review focuses on the preclinical and clinical evidence supporting a potential analgesic role for TTX. In addition, the contribution of specific TTX-sensitive VGSCs to pain is reviewed.
PMCID: PMC3296997  PMID: 22412801
tetrodotoxin; TTX; TTX-sensitive voltage-gated sodium channels; pain; neuropathic pain
16.  479 A 3 Year-old Child with Specific Antibody Deficiency and Allergic Rhinitis. A Case Report 
Specific antibody deficiency (SAD) is a humoral immunodeficiency characterized by normal levels of IgG, IgA, IgM and IgG subclasses but a failure to polysaccharide antigens, manifested with recurrent bacterial respiratory infections. To establish the SAD diagnosis an inadequate IgG antibody response to more than 50% of pneumococcal serotypes after unconjugated pneumococcal immunization are needed. An adequate response is defined as a post-immunization titre of ≥1.3 μg/mL or ≥4 times the preimmunization value.1,2
The record of 1 patient was review and relevant clinical data was collected. A review of the literature about SAD was made.
A 4-year old male with family history of atopic disease, esophageal reflux at 3-months, he began with recurrent upper respiratory tract infections at 1-year old, 1 to 2 events per month, fever (39–40° C), persistent cough and hyaline rhinorrhea, nasal itching and sneezing he was treated with multiple antibiotics, inhaled and oral corticosteroids with mild clinical recovery between episodes. A normal blood cell count and normal levels of IgG 1219 mg/dL, IgA 146 mg/dL, IgM 98 mg/dL and IgG subclasses were determined. Allergic rhinitis and asthma were diagnosed at 3-years old, percutaneous prick skin test was positive to Dermatophagoides farinae, Salsola pestifer, Phleum pratense, Heliantus sp. and specific immunotherapy was started. Despite of treatment he continued with recurrent infections so specific antibody response to polysaccharide pneumococcal antigens was evaluated, he responded less than 50% to 14 pneumococcal serotypes after 23-valent unconjugated pneumococcal vaccine, so SAD was diagnosed and treated with prophylactic antibiotic, pneumococcal polysaccharide conjugated vaccine (10-valent) and specific immunotherapy. He showed clinical improvement, with few mild infections, and controlled rhinitis and asthma.
There are several Primary Immunodeficiency Diseases related to allergic diseases as IgA deficiency and SAD. In the atopic patient that does not improve in spite of specific immunotherapy further investigations are needed to exclude them.
PMCID: PMC3512720
17.  Predictors of School Dropout among Adolescents in Puerto Rico 
This research aims to understand the circumstances associated with school dropout in a cohort of Puerto Rican adolescents.
The study collected data from adolescents and their parents. Information related to school dropout among adolescents was obtained from the second year follow-up data from the longitudinal study funded by NIDA “Risky Families Embedded in Risky Environments” (Grant No. RO1 DA 15301). Data was collected employing a self-administered and a face-to-face interview protocol. Prediction of school dropout was assessed throughout adolescent characteristics, family background, school experiences and behaviors.
During the second follow-up, two years after the baseline assessment, approximately 6.2% of the adolescents reported dropping out from school. Logistic regression analysis indicates that older adolescents (OR=6.6, 1.37-31.67), whose mother used drugs during pregnancy (OR=4.9, 1.31-17.91), who reported high rates of absenteeism (OR=4.8, 1.63-14.13), high school grade retention (OR=3.7, 1.14-12.05), and attended school where teachers were attacked or wounded by students (OR=7.0, 1.44-34.17) were more likely to dropout of school.
These findings emphasize the need to further understand the effects of different elements of adolescents' environment such as family and school. It has been posited that dropping out of school is a process whose characteristics can be detected long before it occurs. The fact that students who dropout are more likely to report skip classes and grade retention can be relevant elements in prevention and early intervention for teachers and other school personnel.
PMCID: PMC2796054  PMID: 19999238
Puerto Rico; adolescents; dropout; school; parents; Hispanic
18.  The Catalytic Subunit of Protein Phosphatase 1 Gamma Regulates Thrombin-Induced Murine Platelet αIIbβ3 Function 
PLoS ONE  2009;4(12):e8304.
Hemostasis and thrombosis are regulated by agonist-induced activation of platelet integrin αIIbβ3. Integrin activation, in turn is mediated by cellular signaling via protein kinases and protein phosphatases. Although the catalytic subunit of protein phosphatase 1 (PP1c) interacts with αIIbβ3, the role of PP1c in platelet reactivity is unclear.
Methodology/Principal Findings
Using γ isoform of PP1c deficient mice (PP1cγ−/−), we show that the platelets have moderately decreased soluble fibrinogen binding and aggregation to low concentrations of thrombin or protease-activated receptor 4 (PAR4)-activating peptide but not to adenosine diphosphate (ADP), collagen or collagen-related peptide (CRP). Thrombin-stimulated PP1cγ−/− platelets showed decreased αIIbβ3 activation despite comparable levels of αIIbβ3, PAR3, PAR4 expression and normal granule secretion. Functions regulated by outside-in integrin αIIbβ3 signaling like adhesion to immobilized fibrinogen and clot retraction were not altered in PP1cγ−/− platelets. Thrombus formation induced by a light/dye injury in the cremaster muscle venules was significantly delayed in PP1cγ−/− mice. Phosphorylation of glycogen synthase kinase (GSK3)β-serine 9 that promotes platelet function, was reduced in thrombin-stimulated PP1cγ−/− platelets by an AKT independent mechanism. Inhibition of GSK3β partially abolished the difference in fibrinogen binding between thrombin-stimulated wild type and PP1cγ−/− platelets.
These studies illustrate a role for PP1cγ in maintaining GSK3β-serine9 phosphorylation downstream of thrombin signaling and promoting thrombus formation via fibrinogen binding and platelet aggregation.
PMCID: PMC2788699  PMID: 20016849
19.  Extracellular Tumor-Related mRNA in Plasma of Lymphoma Patients and Survival Implications 
PLoS ONE  2009;4(12):e8173.
We studied anomalous extracellular mRNAs in plasma from patients with diffuse large B-cell lymphoma (DLBCL) and their survival implications. mRNAs studied have been reported in the literature as markers of poor (BCL2, CCND2, MYC) and favorable outcome (LMO2, BCL6, FN1) in tumors. These markers were also analyzed in lymphoma tissues to test possible associations with their presence in plasma.
Methodology/Principal Findings
mRNA from 42 plasma samples and 12 tumors from patients with DLBCL was analyzed by real-time PCR. Samples post-treatment were studied. The immunohistochemistry of BCL2 and BCL6 was defined. Presence of circulating tumor cells was determined by analyzing the clonality of the immunoglobulin heavy-chain genes by PCR. In DLBCL, MYC mRNA was associated with short overall survival. mRNA targets with unfavorable outcome in tumors were associated with characteristics indicative of poor prognosis, with partial treatment response and with short progression-free survival in patients with complete response. In patients with low IPI score, unfavorable mRNA targets were related to shorter overall survival, partial response, high LDH levels and death. mRNA disappeared in post-treatment samples of patients with complete response, and persisted in those with partial response or death. No associations were found between circulating tumor cells and plasma mRNA. Absence of BCL6 protein in tumors was associated with presence of unfavorable plasma mRNA.
Through a non-invasive procedure, tumor-derived mRNAs can be obtained in plasma. mRNA detected in plasma did not proceed from circulating tumor cells. In our study, unfavorable targets in plasma were associated with poor prognosis in B-cell lymphomas, mainly MYC mRNA. Moreover, the unfavorable targets in plasma could help us to classify patients with poor outcome within the good prognosis group according to IPI.
PMCID: PMC2788245  PMID: 20016842
20.  Platelet glycoprotein Ibα forms catch bonds with human WT vWF but not with type 2B von Willebrand disease vWF 
The Journal of Clinical Investigation  2008;118(9):3195-3207.
Arterial blood flow enhances glycoprotein Ibα (GPIbα) binding to vWF, which initiates platelet adhesion to injured vessels. Mutations in the vWF A1 domain that cause type 2B von Willebrand disease (vWD) reduce the flow requirement for adhesion. Here we show that increasing force on GPIbα/vWF bonds first prolonged (“catch”) and then shortened (“slip”) bond lifetimes. Two type 2B vWD A1 domain mutants, R1306Q and R1450E, converted catch bonds to slip bonds by prolonging bond lifetimes at low forces. Steered molecular dynamics simulations of GPIbα dissociating from the A1 domain suggested mechanisms for catch bonds and their conversion by the A1 domain mutations. Catch bonds caused platelets and GPIbα-coated microspheres to roll more slowly on WT vWF and WT A1 domains as flow increased from suboptimal levels, explaining flow-enhanced rolling. Longer bond lifetimes at low forces eliminated the flow requirement for rolling on R1306Q and R1450E mutant A1 domains. Flowing platelets agglutinated with microspheres bearing R1306Q or R1450E mutant A1 domains, but not WT A1 domains. Therefore, catch bonds may prevent vWF multimers from agglutinating platelets. A disintegrin and metalloproteinase with a thrombospondin type 1 motif–13 (ADAMTS-13) reduced platelet agglutination with microspheres bearing a tridomain A1A2A3 vWF fragment with the R1450E mutation in a shear-dependent manner. We conclude that in type 2B vWD, prolonged lifetimes of vWF bonds with GPIbα on circulating platelets may allow ADAMTS-13 to deplete large vWF multimers, causing bleeding.
PMCID: PMC2518822  PMID: 18725999
21.  Proteomic profiling of neuromas reveals alterations in protein composition and local protein synthesis in hyper-excitable nerves 
Molecular Pain  2008;4:33.
Neuropathic pain may arise following peripheral nerve injury though the molecular mechanisms associated with this are unclear. We used proteomic profiling to examine changes in protein expression associated with the formation of hyper-excitable neuromas derived from rodent saphenous nerves. A two-dimensional difference gel electrophoresis (2D-DIGE) profiling strategy was employed to examine protein expression changes between developing neuromas and normal nerves in whole tissue lysates. We found around 200 proteins which displayed a >1.75-fold change in expression between neuroma and normal nerve and identified 55 of these proteins using mass spectrometry. We also used immunoblotting to examine the expression of low-abundance ion channels Nav1.3, Nav1.8 and calcium channel α2δ-1 subunit in this model, since they have previously been implicated in neuronal hyperexcitability associated with neuropathic pain. Finally, S35methionine in vitro labelling of neuroma and control samples was used to demonstrate local protein synthesis of neuron-specific genes. A number of cytoskeletal proteins, enzymes and proteins associated with oxidative stress were up-regulated in neuromas, whilst overall levels of voltage-gated ion channel proteins were unaffected. We conclude that altered mRNA levels reported in the somata of damaged DRG neurons do not necessarily reflect levels of altered proteins in hyper-excitable damaged nerve endings. An altered repertoire of protein expression, local protein synthesis and topological re-arrangements of ion channels may all play important roles in neuroma hyper-excitability.
PMCID: PMC2525634  PMID: 18700027
22.  The transcription of MGAT4A glycosyl transferase is increased in white cells of peripheral blood of Type 2 Diabetes patients 
BMC Genetics  2007;8:73.
Human glycosylase IV is involved in GLUT2 transporter regulation in pancreatic β cells. A KO of this gene along with a high fat diet in a mice model has been associated with the development of type 2 diabetes (T2D). The aims of this study were to measure and compare the MGAT4A mRNA levels in white blood cells (WBC) from T2D subjects and healthy subjects (T2NB), and to measure the half-life of the MGAT4A mRNA.
We studied a sample of 73 individuals, 40 T2D subjects and 33 T2NB subjects. Anthropometrical and biochemical profiles were registered. The MGAT4A mRNA levels in WBC and the transcript half-life in Jurkat T cells were determined by Real-Time PCR. A blood differential cell counting was made for each individual. Cell counting showed T2D subjects exhibited an increased number of WBC compared to T2NB subjects (P = 0.0001). Biochemical parameters such as fasting glucose (P = 0.0001), and triglycerides (P = 0.002) were statistically significant. T2D subjects had 4.2-fold more MGAT4A transcript compared to T2NB subjects (P = 0.002). The MGAT4A mRNA had a half-life of 2.04 h in Jurkat T cells.
The results of this work suggest that in T2D subjects, high levels of glucose and triglycerides are accompanied by an increase on MGAT4A mRNA levels and WBC count; condition that suggests a pro-inflammatory state due to a chronic metabolic stress.
PMCID: PMC2148049  PMID: 17953760
23.  Oral Candida Isolates Colonizing or Infecting Human Immunodeficiency Virus-Infected and Healthy Persons in Mexico 
Journal of Clinical Microbiology  2005;43(8):4159-4162.
Oral yeast carriage was studied in 312 Mexican subjects. Candida albicans was the most frequent species, but other Candida spp. were isolated from 16.5 to 38.5% of patients. Colonization did not correlate with CD4+ number or viral load, but highly active antiretroviral therapy reduced the frequency of candidiasis. Most isolates were susceptible to fluconazole, but 10.8% were resistant to one or more azoles.
PMCID: PMC1233912  PMID: 16081965
24.  Localization of the Adhesion Receptor Glycoprotein Ib-IX-V Complex to Lipid Rafts Is Required for Platelet Adhesion and Activation 
The Journal of Experimental Medicine  2002;196(8):1057-1066.
The platelet glycoprotein (GP) Ib-IX-V complex mediates the attachment of platelets to the blood vessel wall by binding von Willebrand factor (VWF), an interaction that also transmits signals for platelet activation and aggregation. Because the complex is extensively palmitoylated, a modification known to target proteins to lipid rafts, we investigated the role of raft localization in GP Ib-IX-V functions. In unstimulated platelets, a minor portion of the complex localized to Triton-insoluble raft fractions; this portion increased three to sixfold with platelet activation by VWF. Raft-associated GP Ib-IX-V was selectively palmitoylated, with GP Ib-IX-V–associated palmitate increasing in the raft fraction on VWF-mediated activation. The raft fraction was also the site of association between GP Ib-IX-V and the Fc receptor FcγRIIA. The importance of this association was demonstrated by the ability of the FcγRIIA antibody IV.3 to inhibit shear-induced platelet aggregation. Disruption of rafts by depleting membrane cholesterol impaired several GP Ib-IX-V–dependent platelet fractions: aggregation to VWF under static conditions and under shear stress, tyrosine phosphorylation, and adhesion to a VWF surface. Partial restoration of membrane cholesterol content partially restored shear-induced platelet aggregation and tyrosine phosphorylation. Thus, localization of the GP Ib-IX-V complex within rafts is crucial for both platelet adhesion and postadhesion signaling.
PMCID: PMC2194038  PMID: 12391017
platelets; membrane microdomains; von Willebrand factor; cholesterol; signaling
25.  Immunoproteasome Assembly: Cooperative Incorporation of Interferon γ (IFN-γ)–inducible Subunits  
LMP2, LMP7, and MECL are interferon γ–inducible catalytic subunits of vertebrate 20S proteasomes, which can replace constitutive catalytic subunits (delta, X, and Z, respectively) during proteasome biogenesis. We demonstrate that MECL requires LMP2 for efficient incorporation into preproteasomes, and preproteasomes containing LMP2 and MECL require LMP7 for efficient maturation. The latter effect depends on the presequence of LMP7, but not on LMP7 catalytic activity. This cooperative mechanism favors the assembly of homogeneous “immunoproteasomes” containing all three inducible subunits, suggesting that these subunits act in concert to enhance proteasomal generation of major histocompatibility complex class I–binding peptides.
PMCID: PMC2199179  PMID: 9419215

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